A transgene construct for expressing a heterologous polypeptide in a host cell, seed or organism usually comprises a promoter operably linked to a nucleic acid encoding a heterologous polypeptide. However, transgene expression can be toxic to the host cell, seed or organism, or inhibit growth of the host cell, seed or organism. Furthermore, in the case of transgenic plants, transgenic crop seed can contaminate non-transgenic seed, causing the appearance of an unwanted heterologous polypeptide in a crop. One solution to such problems has been to provide transgene constructs comprising an inducible promoter. In a host cell, cell culture, or organism harboring a transgene comprising an inducible promoter controlling a transgene, expression of the transgene can be delayed until the host cell, cell culture, or organism reaches a predetermined condition or stage of development, size, or cell density. Transcription of the transgene is then induced using a stimulus appropriate for the promoter. However, a significant problem with the use of an inducible promoter to direct transgene expression is that an inducible promoter can support background levels of transgene transcription (i.e., are “leaky”) in the absence of the stimulus. Thus, the host cell comprises RNA coding for the heterologous polypeptide, even in the absence of the stimulus. This RNA can be translated, resulting in background levels of heterologous polypeptide. For example, in host cells comprising a recombinant inducible promoter, the molar concentration ratio of a heterologous polypeptide in an induced host cell compared to an uninduced host cell, can be, for example, about 10:1. Expression of a transgene due to leaky transcription from an inducible promoter can lead to the same kinds of problems encountered when a constitutively active promoter is used. Therefore, it would be useful to provide a system for transgene expression in which the level of expression of the transgene in an unstimulated cell is not measurably greater than in a non-transgenic cell of the same type.
RNA viruses, nucleic acids thereof, and DNA copies of RNA viral sequences have been used to control expression of transgenes. RNA viruses can comprise single-stranded RNA or double-stranded RNA. Single-stranded RNA viruses are either “positive-strand” or “negative-strand” RNA viruses. A positive-strand single-stranded RNA virus comprises sequence in the same reading sense as viral mRNA(s). With the exception of positive strand single-stranded RNA retroviruses, which use a DNA intermediate, replication and transcription of a positive-strand RNA virus involves synthesis of a complementary (negative strand) RNA copy of the viral genome. Synthesis of a complementary RNA copy requires an RNA-dependent RNA polymerase (RDRP) encoded within the viral genome. Viral replication in a cell involves a replication complex comprising the RDRP. During the replication process, the replication complex binds to the 3′ untranslated region (3′ UTR) of the viral RNA, and initiates synthesis of the complementary strand (van Rossum, C. M. A., et al., J. Gen. Virol. 78: 3045-3049, 1997).
U.S. Pat. No. 6,433,248 B1 to Lommel et al. discloses a method of activating transcription of an RNA of interest in a cell. The disclosed method includes the steps of (a) providing a host cell containing a heterologous construct, the heterologous construct comprising an RNA virus subgenomic promoter operatively associated with a heterologous RNA of interest, wherein the promoter does not initiate transcription of the heterologous RNA in the absence of a corresponding RNA virus trans-activating RNA segment, and where the RNA virus trans-activating RNA segment is absent from the host cell; and (b) introducing a trans-activating nucleic acid segment into the host cell so that transcription of the heterologous RNA is initiated. The method relies on the use of a viral trans-activating RNA segment, and because of the presence of RNA comprising coding sequence for the heterologous polypeptide in an uninduced host, is still subject to background levels of expression. Furthermore, unlike the present disclosure, the method does not utilize an RNA sequence complementary to an internal ribosome entry site.
U.S. Pat. No. 6,462,255 B1 to Thurpen discloses high level expression of foreign genes in plants using viral replicons, wherein the replicons code for at least one foreign gene and possess sequences required in cis for replication. Unlike the present disclosure, because of the presence of RNA comprising coding sequence for the heterologous polypeptide in an uninduced host, the methods described are still subject to background levels of expression. Furthermore, the patent does not disclose an RNA comprising an antisense coding sequence, and anti-IRES, and a 3′ UTR as set forth in the present disclosure.
U.S. Pat. No. 6,326,480 B1 to Kovelman et al. discloses a reporter system for assaying positive strand RNA virus replication. The invention describes antisense reporter plasmids comprising a promoter operably linked to a DNA sequence encoding: (a) a sequence complementary to the 3′ end of a viral genome; (b) a reporter gene in antisense orientation; and (c) a sequence complementary to the 5′ end of the viral genome. The patent further describes antisense reporter mRNAs encoding: (a) a sequence complementary to a 3′ end of a viral genome; (b) a reporter gene in antisense orientation; and (c) a sequence complementary to a 5′ end of the viral genome. Unlike the present disclosure, this patent does not disclose a recombinant RNA comprising a sequence complementary to the coding sequence of a heterologous polypeptide, a sequence complementary to an internal ribosome entry site, and a viral 3′ untranslated region, nor does it describe a recombinant mRNA molecule comprising an internal ribosome binding site operably linked to an RNA sequence encoding a heterologous polypeptide and the complement of an internal ribosome entry site.
US Patent Application Publication US 2002/0138873 A1 to Lewandowski et al. discloses a multiple component RNA vector system, consisting of an RNA replicon comprising a 5′ non-translated region, an open reading frame (ORF) homologous to an ORF of an intact or fragments of a non-structural protein of an RNA virus, a sequence non-native to the RNA virus, and a 3′ non-translated region. The recombinant RNA molecules of the present invention do not require an open reading frame (ORF) homologous to an ORF of an intact or fragments of a non-structural protein of an RNA virus or a 5′ non-translated region.
Powell et al. (Proc. Natl. Acad. Sci. USA 86: 6949-6952, 1989) disclosed transgenic tobacco plants that express RNA sequences complementary to the tobacco mosaic virus coat protein coding sequence comprising a tRNA-like structure at the 3′ end of the TMV RNA. Transgenic plants that expressed RNA sequences complementary to the coat protein coding region and the 3′ untranslated region, including the tRNA-like sequences, when challenged with TMV, were protected from infection at low levels of inoculum. These findings did not disclose synthesis or expression of an RNA comprising the complement of a 3′ UTR, an IRES and coding sequence of a heterologous polypeptide.
Zaccomer et al (Gene 136: 87-94, 1993) reported experiments with transgenic rapeseed (Brassica napus) in which the transgenes comprised either a sense or antisense coding sequence of a chloramphenicol acetyltransferase (CAT) gene upstream from a positive strand 3′-terminal 100 nucleotides of the noncoding region of the turnip yellow mosaic virus. RNA complementary to the initial transcript was detected after infection of a host transgenic plant with turnip yellow mosaic virus.
These findings did not disclose synthesis or expression of an RNA comprising the complement of a 3′ UTR, an IRES and coding sequence of a heterologous polypeptide.
Teycheney et al (J. Gen. Virol. 81: 1121-1126, 2000) reported that transcripts of transgenes comprising the 3′ UTR of Lettuce mosaic virus could serve as template for synthesis of complementary negative strand RNA following infection of host tobacco plants with Tobacco etch virus, Tobacco vein mottle virus or Pepper mottle virus, but not with Cucumber mosaic virus. These workers also showed that deletion of the 3′ UTR from the transgene abolished the synthesis of negative strands. These findings did not disclose synthesis or expression of an RNA comprising the complement of a 3′ UTR, an IRES and coding sequence of a heterologous polypeptide.
Therefore, provision of a recombinant RNA comprising a sequence complementary to the coding sequence of a heterologous polypeptide (an “anti-sense coding sequence”), a sequence complementary to an internal ribosome entry site (an “anti-IRES”), and a viral 3′ untranslated region (3′ UTR) as set forth herein, and which is not expected to provide sense strand coding sequence for a heterologous polypeptide prior to stimulation or activation of synthesis of a complementary strand of the recombinant RNA, has not been reported or suggested heretofore.